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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Bee Research Laboratory » Research » Publications at this Location » Publication #252577
Title: Sampling and RNA quality for successful diagnostics using quantitative PCR

Author
item DAINAT, BENJAMIN - Swiss Bee Research Center
item Evans, Jay
item Chen, Yanping - Judy
item NEUMANN, PETER - Swiss Bee Research Center

Submitted to: Journal of Pest Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2010
Publication Date: 5/15/2011
Citation: Dainat, B., Evans, J.D., Chen, Y., Neumann, P. 2011. Sampling and RNA quality for successful diagnostics using quantitative PCR. Journal of Pest Science. 174:150-152.

Interpretive Summary: Honey bees are critical for pollination and there are worldwide efforts to determine the causes and control of honey bee disease. Many of the diagnostic tools emerging from these searches are based on analyses of honey bee RNA, and techniques are improving for RNA extraction and analysis. Nevertheless, these assays require beekeepers and regulators to collect and ship field samples of bees, and these steps are cumbersome and expensive currently. Here we show that bees need not be held at low temperatures or in expensive buffers in order to provide suitable samples for RNA diagnostics of viruses. These results can facilitate research and regulatory efforts aimed at identifying and stopping honey bee disease threats.

Technical Abstract: Diagnostic analyses of RNA targets are widely used in honey bee pathology. These diagnostics can be compromised by the actions of endogenous RNA-degrading enzymes activated upon bee death. RNA degradation can be minimized by storage at ultra-cold temperatures or by immersion in high-salt buffers. However, these methods are not always available in the field or are costly, driving a search for alternate ways to store and transport bees for RNA analyses. While the impact of storage conditions on RNA integrity has been evaluated, the tolerance of standard RT-qPCR diagnostics of honey bee pathogens, given non-ideal collection and storage, has not yet been determined. Given the short regions of RNA now being screened for pathogen diagnostics (generally amplified regions of 100-200 nucleotides), it is conceivable that even degraded RNA may provide a template for precise diagnostics. Here, we evaluated for the first time the impact of the two most convenient sample storage and handling methods (+4°C and Room Temperature) for RT-qPCR honey bee virus diagnostics. Our motivation was to streamline the methods needed to collect, transport, and store honey bee samples destined for pathogen diagnostics. We show that samples held at room temperature for times anticipated for sample transport are in fact suitable for virus diagnostics. Our data will be useful for the standardization of sampling methods across countries and laboratories.